Methods and compositions for multiplex pcr

Abstract

The present invention provides methods, compositions, kits, systems and apparatus that are useful for determining copy number variation of one or more nucleic acids present in a sample. In some aspects, the method includes various target-specific primers that allow for the selective amplification of one or more target nucleic acids in the sample. In yet another aspect, the invention relates to determining copy number variation with respect to gene or chromosome representation of a nucleic acid in the sample. In some aspects, the method for determining copy number variation of different target nucleic acids in a sample using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including diagnosis, predictive therapeutic regimes or other therapeutic purposes.

1 . A method for determining copy number variation, comprising amplifying a plurality of different target sequences in a sample, comprising:
 a) producing a plurality of different amplified target sequences within a single amplification reaction mixture by contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, where at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group, and wherein the amplifying includes no more than one round of target specific selection for at least one of the target sequences to be amplified;   b) cleaving a cleavable group from at least one amplified target sequence;   c) producing one or more adapter-ligated amplified target sequences, by ligating at least one adapter to at least one amplified target sequence;   d) reamplifying the at least one adapter-ligated amplified target sequence using primers;   e) sequencing the at least one amplified adaptor-ligated target sequence;   f) calculating the number of sequencing reads for the at least one amplified adaptor-ligated target sequence; and,   g) determining copy number variation of the at least one amplified adaptor-ligated target sequence.   
     
     
         2 . The method of  claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a chromosomal or gene duplication. 
     
     
         3 . The method of  claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a chromosomal or gene deletion. 
     
     
         4 . The method of  claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a loss of heterozygosity. 
     
     
         5 . The method of  claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a copy number variation associated with cancer. 
     
     
         6 . The method of  claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a copy number variation associated with an inherited disease. 
     
     
         7 . The method of  claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a copy number variation associated with an aneuploidy. 
     
     
         8 . The method of  claim 7 , wherein the aneuploidy is a sex chromosome aneuploidy. 
     
     
         9 . The method of  claim 8 , wherein the aneuploidy is selected from the group consisting of XO, XXX, XXXX, XXXXX, XXY, XXYY, XXXY, XXYYY, XXXYY, XXXXY, XYY, XYYY or XYYYY. 
     
     
         10 . A method for determining copy number variation, comprising amplifying a plurality of different target sequences in two or more samples, comprising:
 a) producing a plurality of different amplified target sequences within a single amplification reaction mixture by contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, where at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group, and wherein the amplifying includes no more than one round of target specific selection for at least one of the target sequences to be amplified;   b) cleaving a cleavable group from at least one amplified target sequence;   c) producing one or more barcode adapter-ligated amplified target sequences, by ligating at least one different barcode adapter to at least one amplified target sequence from each sample;   d) reamplifying the at least one barcoded adapter-ligated amplified target sequence from each sample using primers;   e) sequencing the at least one amplified adaptor-ligated target sequence from each sample;   f) calculating the number of sequencing reads for the at least one amplified adaptor-ligated target sequence from each sample; and,   g) determining copy number variation of the at least one amplified adaptor-ligated target sequence for each sample.   
     
     
         11 . The method of  claim 10 , wherein the calculating comprises determining the total number of mapped sequencing reads of an amplified adaptor-ligated target sequence divided by the total number of mapped sequencing reads. 
     
     
         12 . The method of  claim 10 , wherein the calculating comprises determining the percent ratio of the amplified adaptor-ligated target sequence. 
     
     
         13 . The method of  claim 10 , wherein the calculating comprises determining the percent frequency of the amplified adaptor-ligated target sequence. 
     
     
         14 . The method of  claim 10 , wherein the calculating comprises determining the log 2  ratio of the amplified adaptor-ligated target sequence. 
     
     
         15 . The method of  claim 10 , wherein determining copy number variation comprises an over-representation of at least one amplified adaptor-ligated target sequence associated with a disorder or disease. 
     
     
         16 . The method of  claim 10 , wherein determining copy number variation comprises an under-representation of at least one amplified adaptor-ligated target sequence associated with a disorder or disease.